All adherent cells were maintained in DMEM/F12 medium supplemented with 10% (v/v) fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO2. Spheroids were generated from cells after plating at a density of 1000 cells/well in ultra-low attachment 6-well culture plates (Corning, NY, USA). Spontaneously generated spheroids were cultured in serum-free DMEM/F12 medium supplemented with 2% B-27 Supplement (Invitrogen, Carlsbad, CA, USA) without vitamin A, 20 ng/mL basic fibroblast growth factor (FGF, Peprotech, Rocky Hill, NJ, USA), 20 ng/mL epidermal growth factor (EGF, Peprotech), 10 ng/mL leukemia inhibitory factor (LIF, Peprotech) and insulin-transferrinselenium (ITS, Invitrogen). Fresh medium was added every 3 days, and spheroids were cultured for approximately 2 weeks before they reached a diameter of approximately 150 μm. The spheres were collected by gentle centrifugation, dissociated with accutase (Invitrogen) and mechanically disrupted with a pipette. The resulting single-cell suspension was then centrifuged and resuspended in serum-free medium to allow reforming of spheres.

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